Comp. lmmun. Microbiol. infect. Dis. Vol. 14, No. 2, pp, 151-163, 1991 Printed in Great Britain. All rights reserved

0147-9571/91 $3.00+0.00 Copyright © 1991 Pergamon Press plc

MOLECULAR BIOLOGY OF PSEUDORABIES (AUJESZKY'S DISEASE) VIRUS THOMAS C. METTENLEITER Federal Research Centre for Virus Diseases of Animals, P.O. Box 1149, D-7400 Tiibingen, Germany Abstract--In this review, some of the aspects concerning the molecular biology of pseudorabies

virus (PrV), the causative agent of Aujeszky's disease, will be discussed. It will mainly focus on new findings concerning viral glycoproteins, factors determining PrV virulence, the problem of PrV latency and the developments regarding genetically engineered vaccines. Key words: herpesvirus, pseudorabies virus, glycoproteins, virulence, latency, vaccines.

BIOLOGIE MOLI~CULAIRE DU VIRUS PSEUDORABIQUE (VIRUS DE LA MALADIE AUJESZKY) R~sum~---Ce rapport discute des questions de biologie molrculaire propre au virus d'Aujeszky. Les nouvelles connaissances sur les glycoprotrines virales, sur les facteurs de la virulence, les probl6mes de la latence du virus et le drveloppement des vaccins produits grnrtiquement sont particulirrement mises en 6vidence. Mots-clef~: virus herpes, virus pseudorabique, glycoprot~ines, virulence, latence, vaccins.

INTRODUCTION

Pseudorabies virus (PrV) is the causative agent of Aujeszky's disease (AD). It belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae [1]. The correct taxonomic name is Suid herpesvirus 1. Aujeszky's disease, originally described in cattle, is now mainly affecting pigs and is widespread in pig farms in Eastern and Central Europe, the U.S.A. and in South-East Asia where it causes heavy economic losses. This review will mainly focus on some of the new developments that have taken place regarding the molecular biology of PrV. Special emphasis will be placed on new findings concerning viral glycoproteins, factors determining virulence, the problem of PrV latency and the recent introduction of genetically engineered vaccines. Due to space constraints the review has to be limited and can not aim at summarizing all interesting work that has been performed on the molecular biology of PrV (for a recent detailed review see Ref. [2]).

VIRAL GLYCOPROTEINS

The genome of PrV consists of approx. 150,000 bp which is sufficient to encode between 70 and 100 viral proteins. It has a very high G + C content of 73%. The genome is divided into two unique portions, the unique long (U1) and unique short (Us) region by inverted repeat segments (IR) ([3]; see Fig. 1). As in all herpesviruses transcription is strictly

151

152

THOMAS C. METTENLEITER

controlled in a cascade-like fashion. The single PrV immediate-early gene is transcribed without need for cellular protein synthesis. Therefore large amounts of IE transcripts accumulate in cells infected with PrV in the presence of cycloheximide [4]. The immediateearly protein is a potent transactivator of early genes [5]. They are characterized by expression before the onset of viral D N A replication, i.e. 1-2 h p.i. To this class belong the genes encoding the major viral DNA-binding protein (DBP) and the thymidine kinase (TK) [3]. Early-late genes also begin to be expressed before viral DNA replication but reach their maximum expression levels after replication had started. Finally, late genes are expressed exclusively after D N A replication had taken place. Viral structural proteins appear to belong mostly to the early-late or late class of viral genes [3]. Whereas complete sequence information is available for some of the human herpesviruses such as Epstein-Barr virus (EBV) [6], herpes simplex virus (HSV) [7, 8], varicellazoster virus (VZV) [9] and human cytomegalovirus (HCMV) [10] only small parts of the PrV genome have been sequenced. Next to about 15 kbp contained mostly in the repeat region [91] the second longest contiguous stretch of D N A that has been sequenced so far (approx. 1 1 kbp) includes the Us region (Fig. l). There, the genes for four of the PrV glycoproteins (gp) have been found [11-14]. In the U~ part three more glycoprotein genes have been localized [15-19]. The seven glycoproteins found in PrV so far have been designated gI, glI, gill, gp63, gp50, gX and gH. Interestingly, all of them mop units

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0.1 I

0.2 I

0.3 l

0.5

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0.7 I

0.6 I

I

0.6 I IR

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1

2 I

0.9 I

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16 9 11~5 & :,,:

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Fig. 1. Locationsof genes mapped in pseudorabiesvirus. (a) Schematicdiagram of the PrV genome. Open boxes represent inverted repeat regions (IR = internal repeat; TR = terminal repeat) that separate the unique long (UI) from the unique short (Us) component. (b) Bam HI-restriction fragment map. (c) Location of genes. Arrows indicate transcriptionaldirection. Glycoproteingenes are highlighted. Genes either mapped (H) or mapped and sequenced 0--~) includethose coding for glI [16], ICP 18.5 [85], DBP (136 K DNA-binding protein [3]), Pol (DNA-polymerase;[3]), gill [15], TK (thymidinekinase; [3]), gH [18, 19], MCP (major capsid protein; [3, 69]), RSp40 [86], PK (protein kinase; [87,88]), gX [14], gp50 154], gp63 [13], gI [11,13], IlK 1891, 28K [88], IEP (immediate-early protein; [91]) (diagram by courtesy of W. Fuchs and R. Straub).

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153

constitute homologs of glycoproteins found in other herpesviruses. Homologies to the known herpes simplex virus (HSV) glycoproteins are listed in Table 1. Of these seven glycoproteins, four have been shown to be nonessential for viral replication in tissue culture, i.e. gI, gill, gp63 and gX [20-23]. The genes encoding glycoproteins glI, gp50 and most likely also that coding for gH cannot be deleted from the viral genome without abolishing viability of the virus and are therefore designated as essential. Whereas most of the glycoproteins are structural components of the mature virion [24, 25], gX is released from infected cells into the medium in large amounts but can not be found in the virion [3, 14]. Viral glycoproteins are important for the interaction of the virus with its host. They not only mediate infection of target cells but are also major antigens recognized by the infected host's immune system. Detailed knowledge about the role the PrV glycoproteins play in both processes has only recently begun to emerge. Analysis of genetically engineered mutants which carry lesions in the gI gene showed that deletion of gI alters the growth characteristics of PrV [26, 27]. Surprisingly, this effect is cell-type specific. Glycoprotein gI-negative PrV mutants show a distinct growth advantage in chicken embryo fibroblasts (CEF) which is due to a more efficient release of mature virions. However, growth of wildtype PrV is clearly favored over gI PrV in rabbit kidney (RK) cells [28-30]. Detailed analyses proved that the functional entity influencing viral growth consists of a complex of two glycoproteins, gI and gp63. Deletion of either component leads to inactivation of the function and to the expected phenotype. The gI/gp63 complex is noncovalently linked and can be demonstrated by immunoprecipitation [29]. Analysis of an attenuated PrV strain containing a lesion in the glycoprotein gIII-gene which leads to low gIII-expression and failure of most of the gIII to be incorporated into mature virions showed the interaction of the gI/gp63 complex with gIII in mediating virus release [26, 27, 31]. Whereas inactivation of either gI/gp63 or gIII has no obvious effect on virus release from RK cells, deletion of gI/gp63 and gIII significantly reduces the ability of the virus to be released from RK cells [30]. Deletion of gIII, while having no effect on release of PrV from RK cells, significantly reduces release from CEF. These results show that three glycoproteins, gI, gp63 and gIII interact in one aspect of viral growth, virus release. They also show that this effect is highly cell-type specific and is therefore dependent on viral and cellular functions. The major role of gIlI, however, is its involvement in the first step of virus infection, attachment of the virus to its host cell. This can be deduced from the fact that complement-independently neutralizing anti-gIII antibodies [24] block virus infection only when present prior to adsorption of virions but not after attachment had taken place Table 1. Glycoprotein-homologies PrV/HSV PrV

Location

Essential

HSV

gI glI gill gp50 gp63 gX gH -? ?

Us UI Ui Us US U~ U~ U~ Ut U~

+ -+ + + +

gE gB gC gD gl gG gH gJ gK gL

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THOMASC. METTENLEITER

Fig. 2). Genetically engineered glII deletion mutants of PrV have then been used to show that gill-negative viruses exhibit a decrease in titer compared to wildtype PrV of approx. 10-100 fold [31, 32]. This is mainly due to an impaired ability of these mutants to adsorb to target cells. The severity of this defect is different in different target cells and appears most prominent in bovine cells (MDBK) [31]. Further analyses showed that glII mediates the attachment step by binding to a cellular receptor containing a heparin-like moiety [33, 34]. Removal of the heparin moiety by heparinase treatment of cells prior to infection considerably reduces the ability of wildtype PrV to adsorb as does absence of gill from the PrV gill virions. However, since g i l l - virions are still infectious, a second, gillindependent mode of adsorption leading to productive infection must exist. It is interesting to note that HSV-1 has also been shown to adsorb via a heparin-containing moiety [35] and this interaction is mediated by the glII-homologous glycoprotein C [36]. However, replacement of PrV-glII with HSV-gC did not lead to functional complementation but rather to unexpected phenotypes such as lack of incorporation of HSV-gC into the PrV envelope [37]. Lack of gill also leads to a slower penetration of virions into target cells [38], a defect that is coupled with the inefficient adsorption [39]. Interestingly both defects can be overcome by the polybasic compound polylysine. Polylysine restores to gill virions the capability for efficient adsorption and it also leads to an accelerated penetration of gill virions into the cell [40]. In summary, the non-essential glycoproteins gI, gp63, and glII are involved in mediating steps at the very beginning (glII) and the end (gI/gp63/glII) of virus replication. In both

100

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Before adsorption

80

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60

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.N 40

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anti-gl anti-gll anti-gill anti-gp50 Fig. 2. Pre- and post-adsorption neutralization. Virus suspensions were incubated with the indicated monoclonalantibodies either before or after adsorption had taken place. Neutralization was assessed as percent plaque reduction. The anti-gI mAb was includedas a negativecontrol since anti-gI mAbs do not exhibit complement-independentneutralizing activity. It is evident that the anti-gill mAb neutralizes only when added to the virus before adsorption, whereas both the anti-glI and anti-gp50 rnAbs were able to etiicientlyneutralize already adsorbed virus.

Molecular biologyof pseudorabies virus

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cases, the severity of the defect is cell-type dependent which supports considerations that herpesviral nonessential glycoproteins are involved in modulating the capability of the virus to infect different target cells. The function of glycoprotein gX, which is non-structural and secreted in large amounts from infected cells, has not yet been elucidated. However, recently a virus mutant has been constructed that lacks all of the four non-essential glycoproteins [41]. The fact that this mutant is viable, at least in cell culture, proves that all functions these glycoproteins fulfil either alone or in combination are either not absolutely required for viral growth or are provided for by other viral or cellular gene products. It is, however, obvious that expression of nonessential glycoproteins is beneficial for the virus under certain conditions. That no field strain has been isolated so far that lacked any of the glycoproteins indicates that survival of the virus in the animal is one of the conditions requiring nonessential glycoproteins. Glycoprotein glI, the most prominent glycoprotein found in purified virions, is represented by a disulfide-linked complex (mol. wt = 155 kDa) of two glycopolypeptides (67 and 58 kDa) that are derived by proteolytic cleavage from a single protein precursor [17, 24, 25, 42]. It belongs to a class of glycoproteins that is the most highly conserved among herpesviruses. The prototype is the gB-glycoprotein of HSV and they are therefore called gB-homologs. The gB of HSV-1 exhibits 50% homology on the amino acid level and 62% homology on the DNA level to PrV-glI [42]. All the gB-homologous proteins are essential for their respective viruses. Therefore, mutational analyses have been difficult. Experiments using a complement-independently neutralizing anti-glI monoclonal antibody showed that this antibody can still block virus infectivity even after the virus has adsorbed to target cells indicating a step such as virus penetration as the process that is inhibited (Fig. 2). The availability of transgenic cell lines that express the gene to be deleted from the viral genome and provide the necessary gene product in t r a n s allowed the isolation of mutants with lesions in essential genes. Recently, two glI-expressing cell lines have been constructed and they were used to isolate a glI-deficient PrV mutant [43]. This virus can only be propagated on complementing cells proving the essential character of the glI-protein. Viruses lacking glI are non-infectious which is due to a defect in viral penetration. Similar results have been shown for the gB of HSV [44]. The high degree of conservation between the gB-homologs already points to a common function. Final proof that the gB-homologous proteins of two different herpesviruses indeed fulfil the same functional role stems from recent data that showed that glI-negative PrV can be fully complemented by cell lines expressing the gB-protein of bovine herpesvirus 1 (BHV-1). The foreign gB becomes incorporated into the PrV envelope and mediates efficient penetration of the pseudotypic virus into host cells [43]. In addition to its function in penetration adsorption studies indicate that glI forms a glycoprotein complex with gill that mediates binding to the heparin-containing surface receptor [33]. However, glI alone does not appear to be able to bind to heparin. Glycoprotein gp50, homolog to HSV gD, with a size of 50-60 kDa lacks N-linked carbohydrates [12]. Its gene is located in the Us region upstream from the gene encoding gp63. It has recently been shown that both proteins are most likely translated from a single mRNA that terminates downstream from the gp63-gene [45]. Preliminary functional analyses showed that gp50 is also involved in a step following virus attachment, probably penetration [39, 46]. This is indicated by the fact that complement-independently neutralizing monoclonal anti-gp50 antibodies are still able to efficiently inactivate virus that has

156

THOMAS C. METTENLEITER

already adsorbed to the target cell (Fig. 2). However, whereas HSV-gD is essential for both initial penetration and cell-cell fusion the PrV-gp50 appears to be essential only for the penetration event [46]. An interesting aspect to note is that cell-lines expressing gp50 have been shown to be partially resistant to superinfection with either PrV or HSV [47]. It has also been reported that cell-lines expressing gD of HSV are partially resistant to HSV superinfection [48]. These results might indicate a common functional role for the gD-homologs of these two viruses in the infectious process. However, experiments designed to test a direct functional equivalency between the gD-homologs, i.e. the capability of gp50 to complement a gD-defect in HSV, did not indicate complementation [49]. It therefore remains to be determined whether gD-homologs of more closely related viruses such as PrV and BHV-I are able to complement defects reciprocally. A gene encoding a PrV glycoprotein homologous to gH of HSV has only recently been sequenced. It resides in the U 1region downstream from the thymidine kinase gene ([18, 19]; see Fig. 1). The location of the gH-gene downstream from the TK-gene is conserved throughout the herpesviruses. Comparison of the amino acid sequences of different gH-homologs shows that gH is, next to gII, the second most highly conserved glycoprotein. Homologies range from over 30% among the alphaherpesviruses PrV, BHV-I and VZV to 19% when PrV-gH is compared to EBV-gH. In PrV a 2.1 kb m R N A has been determined as the gH-specific transcript. This gH-mRNA is translated in vitro into a 72 kDa primary translation product [19]. However, attempts to generate antibodies that recognize mature PrV-gH have failed so far. New data indicate the presence of at least three more glycoproteins in HSV. The gene for a non-essential glycoprotein (gJ) resides in the Us region [50]. A homologous gene has not been found in the Us region of the PrV genome. Two other HSV glycoproteins (gK and gL) are encoded by open reading frames UL53 and ULI, respectively, and are thought to be essential for HSV replication ([51], D. C. Johnson, personal communication). Since the corresponding regions in the PrV genome have not been sequenced so far, no data regarding a possible PrV homolog exist. ROLE OF PrV GLYCOPROTEINS IN THE IMMUNE RESPONSE After infection of animals with PrV neutralizing complement-dependent antibodies can be found as early as 4 d p.i. Complement-independent neutralizing antibody activity is normally not seen before day 10 p.i. and may even take much longer to appear [2]. The availability of monoclonal antibodies directed against different antigens of either purified Pr virions or PrV infected cells allowed a more thorough investigation of the properties of anti-glycoprotein antibodies. It has been shown that antibodies directed against gp50 are the most potent in neutralizing PrV without the aid of complement [52 54]. Passive transfer of anti-gp50 antibodies can protect mice and pigs against a lethal PrV challenge [55, 56]. Vaccination using either lysates of cells expressing gp50 (mice, pigs) or a gp50 expressing vaccinia recombinant (mice) can also protect the animals from a lethal challenge [56]. It is therefore concluded that gp50 is a major target for protective antibodies. Several antibodies have been isolated that are directed against gIII and that are able to neutralize PrV infectivity complement-independently in vitro [24] by inhibiting virus attachment (see above). In studies performed by Ben-Porat and colleagues it was also shown that anti-gIII antibodies represent a major portion of neutralizing antibodies in sera from reconvalescent pigs [57].

Molecular

biology of pseudorabies

virus

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In addition, gill is a major target antigen for murine and swine virus-specific cytotoxic T-lymphocytes [58] and passive transfer of anti-gill mAbs protected mice and pigs from a lethal PrV challenge [52, 59]. Taking these data together it appears that gill is one of the major immunogens of PrV. Unfortunately gill has also been shown to undergo antigenic drift which might enable some viruses to better elude the host's immune defence mechanisms [57]. Complement-independently neutralizing anti-glI monoclonal antibodies have been isolated ([2]; see Fig. 2). Complement-dependent neutralizing antibodies could be isolated that are directed against gI, glI, glII and gp50 (see Table 2). Passive intraperitoneal transfer of both anti-gI and anti-glI antibodies resulted in protection of mice from a lethal PrV challenge [3, 60]. Whereas glI does not show antigenic variation when different field-isolates and laboratory strains were tested by monoclonal antibodies (unpublished), virus strains do show different reactivities with anti-gI mAbs. This is due to a certain degree of antigenic drift [57] as well as differences in the level of expression of gI. The level of gI-expression in a given population of virions from a laboratory strain can vary from undetectable to high level expression [61]. A field-isolate has also been described that expresses an altered gI gene product [61]. However, neutralization studies using monoclonal antibodies and viruses with a defined gI-genotype showed that anti-gI antibodies probably only play a minor role in PrV neutralization in vivo [57]. Monoclonal antibodies against gp63 are available [52]. They do not exhibit PrV-neutralizing activity without complement. The lack of neutralizing activity of anti-gX mAbs both with and without complement is expected since gX is not a constituent of the viral envelope. As mentioned, anti-gH(PrV) antibodies have not been isolated to date. In summary, the data show that gill and gp50 certainly constitute major immunogens of PrV. The relative contribution of the other glycoproteins to an immune response of the infected organism directed against the whole virus is, however, difficult to assess and still largely unclear. VIRULENCE FACTORS Knowledge about the factors that influence virulence of PrV, i.e. its capability to cause disease in, or kill, infected animals, are basic for the understanding ofPrV pathogenesis and for the construction and evaluation of attenuated strains used for vaccination. The first Table 2. Functions o f PrV glycoproteins PrV glycoproteins Function Essential for replication in cell culture Present in virions Neutralization - C Neutralization + C Adsorption Penetration Release Fc-reeeptor Cell-mediated i m m u n i t y

gI

gll

gill

gp63

gp50

gX

gH

No + + +

Yes + + + ( + ):t +

No + + + + ( + )~ +

No + -

Yes + + +

No -

(Yes)* "t

+ N o t found

+

+

*The essential character o f g H o f PrV has not yet been proven but is assumed. f N o entry m e a n s that d a t a are not available. ~glI is complexed with g i l l and as such probably participates in adsorption. §The effect o f g l l I on virus penetration is directly related to its function in adsorption.

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THOMAS C. METTENLEITER

gene identified to be involved in expression of the virulent phenotype in herpesviruses was that coding for the enzyme thymidine kinase (TK). TK-negative PrV mutants exhibit a greatly reduced virulence in different host species [62]. They were selected by growth of wildtype virus stocks in medium containing nucleotide analogs such as arabino-thymidine (ara-T) [63], arabino-cytidine (ara-C), bromodeoxyuridine (BrdU) or iododeoxyuridine (IdU) [64]. Complementation analyses of other attenuated strains as well as the construction of genetically engineered mutants identified other genes or genomic regions involved in virulence. Some attenuated strains that had been obtained by classical ways through multiple passages in embryonated eggs or chicken embryo fibroblasts (CEF) were shown to contain genomic deletions that invariably comprise the gene coding for glycoprotein gI [20, 28, 65]. Further analysis showed that gI indeed plays a role in the expression of PrV virulence [66]. Deletion of gI, however, does not necessarily result in an avirulent phenotype [66, 67]. To decrease virulence under a detectable level another glycoprotein gene, that coding for gIII, had to be inactivated. The resulting gIII /g! mutant is avirulent when tested in either a chicken model system (after intracerebral infection of 1 d old chicks) or in pigs [68]. This again points to a synergistic effect of deletions in glycoprotein genes, since a mutant deficient in gIII-expression only is still virulent for chicken and pigs. Another region of the genome implicated in virulence is contained in the BamHI-fragment 4 which encodes several capsid proteins [69]. The molecular basis for this defect is, however, not known. Careful analysis proved that, e.g. in the vaccine strain Bartha three independent defects are present: (i) A deletion of DNA encompassing the gI- and gp63-genes [65]; (ii) A mutation in the glycoprotein gIII-gene (most likely a signal sequence mutation is responsible for the observed phenotype) [70]; (iii) A mutation in fragment BamHI-4 encompassing some capsid protein genes [69]. Analyses of further vaccine strains points to a number of additional genes or genomic regions that play a role in viral virulence [71]. It can therefore be stated that virulence in PrV is controlled multigenically, i.e. that several genes are involved in the full expression of PrV virulence [71]. LATENCY A characteristic of herpesvirus infections is the ability of the virus to remain in the infected host after the acute phase of infection in a latent state. During latency infectious virus cannot be detected, whereas viral genomes are present. Several exogenous stimuli such as ultraviolet light, fever, stress or certain biologically active compounds, e.g. dexamethasone or prostaglandins are able to reactivate the latent virus. During latency the viral DNA exists mainly in a linear form although circular PrV-DNAs have been demonstrated in several cases [72). Latent DNA can be found in pigs in the trigeminal ganglia, the brain, the spinal chord, the tonsils and in cells of the hematopoietic system, mainly in the bone marrow, by different molecular hybridization techniques or polymerase chain reaction [72, 73]. During latency there appears to be limited transcription from the viral genome [74] giving rise to transcripts that run in anti-sense to the orientation of the immediate-early gene transcript [75]. These RNAs originate from sequences encompassing

Molecular biology of pseudorabies virus

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Barn HI-fragments 6 (partially), 14, 8', 8 and 5 (partially) ([76]; cf. Fig. 1). Whether these transcripts play a role in regulating viral latency by interaction with the immediate-early m R N A or whether they are involved in the transition from latency to a reactivated state as is the case for the HSV latency associated transcripts (LAT) [77, 78] has to be shown. So far, no clear picture of the importance of the LAT of herpesviruses has emerged. It should be emphasized that none of the presently used PrV vaccines is capable of protecting the animal from latency of the challenge virus [79]. Whether prevention from latency can ever be achieved using current vaccination procedures appears doubtful. GENETICALLY ENGINEERED VACCINES One of the major developments in vaccination against AD has been the recent introduction of genetically engineered live vaccine strains. On the basis of results obtained after molecular investigation of classical attenuated PrV strains and the identification of virulence-associated genes modern technology was used to introduce defined lesions into PrV genome. Most genetically engineered vaccines carry a lesion in the thymidine-kinase gene which considerably reduces virulence of the virus [63]. In addition, a serologically identifiable marker deletion comprising the gene for either glycoprotein gI [80], gill [81] or gX [82] had been introduced. This gives the ideal situation that vaccinated animals can be differentiated from field virus infected animals. Animals vaccinated with a so-called "deleted" vaccine are not able to mount an immune response against the protein whose gene has been deleted in the vaccine virus genome. In contrast, wildtype virus-infected animals produce antibodies against all the viral glycoproteins. Differentiating ELISA-tests specific for the deleted marker protein then allow discrimination between infected animals that can be removed and vaccinated animals [83]. On the basis of this screening system in both Europe and the U.S.A. ambitious PrV eradication programs have been started. Among the future prospects in vaccine development the construction of viral vector vaccines based on PrV is noteworthy, vanZijl and colleagues [84] have succeeded in constructing a recombinant PrV strain expressing a major immunogenic glycoprotein of the virus causing classical swine fever, HCV (hog cholera virus). Vaccination with this recombinant virus protected pigs not only from a PrV-challenge but also from a lethal challenge by HCV. A single vaccine virus therefore was able to protect against two important virus diseases in pigs. Several other combinations are conceivable, e.g. introduction of the immunogens of pig rotavirus, transmissible gastroenteritis virus or pig influenza virus into a PrV-based vector vaccine. One major advantage of PrV as a vector as compared to vaccinia virus is its apathogenicity for humans [2]. Whether these vaccines will be accepted by both the public and the registration authorities, however, remains to be seen. In summary, genetically engineered live vaccines against AD have been introduced and they represent the forerunner for probably many others to follow. Both, classical and genetically engineered vaccines contain serological markers which allow discrimination between vaccinated and wildtype infected animals and constitute the basis for newly conceived eradication programs. Acknowledgements--The author gratefullyacknowledgesgift of monoclonalantibodies from T. Ben-Porat, H. J. Rziha and M. W. Wathen and thanks several colleagues for the permission to cite their work prior to publication.

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